Diazepam-insensitive [3H]Ro 15-4513 binding in intact cultured cerebellar granule cells

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Abstract

[3H]Ro 15-4513, a partial inverse agonist at the benzodiazepine receptor, binds to two sites in the rat cerebellum, only one of which is sensitive to diazepam. The diazepam-insensitive component, whose identity is unknown, is unique to this brain area. We studied the binding of [3H]Ro 15-4513 in cultured cerebellar granule cells to characterize its binding sites in a specified neuronal cell population and to determine the effects of ethanol on the binding. We also compared the properties of [3H]Ro 15-4513 binding in washed membranes of cultured cells and 14-day-old rat cerebella. [3H]Ro 15-4513 had two binding components in intact granule cells, one sensitive to diazepam, that probably represents binding to the benzodiazepine agonist site, the other sensitive to an antagonist (Ro 15-1788) and two inverse agonists (ethyl-β-carboline-3-carboxylate, βCCE and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, DMCM) of the benzodiazepine receptor (diazepam-insensitive binding). This diazepam-insensitive] binding was stimulated by GABA; the maximal increase in binding was about 60% with an EC50 of 0.3 μM. The effect of GABA (10 μM) on the diazepam-insensitive binding was unaffected by 100 μM nipecotic acid but was partially inhibited by 100 μM bicuculline. The stimulation by GABA was also seen at 37°C with washed membranes of 14-day-old rat whole cerebella in the presence of micromolar diazepam. The diazepam-insensitive [3H]Ro 15-4513 binding was inhibited by picrotoxin, TBOB and DMCM (all 10 μM). Ethanol decreased the diazepam-sensitive component moderately in intact granule cells, e.g. ca. 35% decrease at 100 mM ethanol. The diazepam-insensitive [3H]Ro 15-4513 binding apparently was affected only at very high non-physiological ethanol concentrations. The diazepam-insensitive [3H]Ro 15-4513 binding site may be associated with both GABAA and benzodiazepine receptors in the cerebellar granule cells. Its physiological role as a possible mediator of the actions of benzodiazepine inverse agonists remains to be studied.

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